ABSTRACT
Objective To investigate the effects of unaggregated amyloid ? protein(A?25～35) on transient outward potassium channel(IA) in Rat Hippocampal CA3 Pyramidal Neurons.Methods Patch-clamp technique with whole cell recording was used.Results Unaggregated A?25～35 inhibited IA in neonatal rat hippocampal CA3 pyramidal neurons and displayed a time-,concentration- and voltage-dependent manner;the dynamic characteristics of IA were influenced:shifted the steady-state activation and inactivation curves to left significantly.Conclusion These results suggest that the inhibition of unaggregated A?25-35 on transient outward potassium channel in acutely isolated hippocampal CA3 pyramidal neurons may be an important mechanism of its toxicity,which participates in pathological changes of AD.
ABSTRACT
Objective To study the effects of A?25-35 on the expression of gene P21,CDK4,E2F1 and BAX in cultured PC12 cells.MethodsPC12 cells were treated with 25 ?mol/L A?25-35,the relation between cell cycle redistribution and apoptosis was analyzed by flow cytometry(FCM).Protein and mRNA expression of P21,CDK4,E2F1 and BAX were detected by RT-PCR and Western-blot respectively.Results About 90% PC12 cells were found arrest on G0/G1 by FCM being deprived serum.Treated with 25 ?mol/L A?25-35 for 8,16,24 h,the percent of S phase cells was raised remarkably(P
ABSTRACT
Objective To examine the effect and mechanism of sodium ferulate inhibiting P38 MAPK in macrophage on neurotoxicity which was mediated by fribrilar-amyloid-induced.Methods The isolated peritoneal macropohages were cultured.P38 MAPK protein in nuclear extracts was analyzed by Western blot and production of tumor necrosis factor-?(TNF-?) was detected by ELISA.For neuron-macrophage co-cultures,Microtubute-associated protein-2(MAP-2) expression was detected by immunocytochemical technique.The level of LDH in the medium was measured.Results The production of TNF-? and P38 MAPK protein in nuclear extracts increased significantly by incubation with A?25-35.LDH efflux in neuron-macrophage co-culture medium increased and MAP-2 expression decreased significantly(P
ABSTRACT
Objective To investigate the effect of catalpol from Radix Rehmanniae on A?25-35-induced apoptosis of PC12 cells.Methods PC12 cells were routinely cultivated and treated by A?25-35(final concentration,20 ?mol/L) 24 hours after the addition of catalpol or saline.Forty-eight hours later,cells were examined for viability and apoptosis by MTT method and TUNEL method,respectively,while Bax and Bcl-2 mRNA expression were analyzed by semi-quantitive RT-PCR. Results Catalpol could significantly elevate the viability at 1?10-5 mol/L and 1?10-4 mol/L(P
ABSTRACT
Objective To study the effect of procyanidins (PC) on mRNA and protein expression of par-4 and bcl-2 genes in PC12 cells induced by A?_ 25-35 . Methods Cell survival rate was evaluated by MTT assay and apoptosis was analyzed by Hoechst 33258-PI fluorescence staining. The expressions of mRNA and protein for par-4 and bcl-2 were tested by RT-PCR and Western blotting. Results Pretreatment with different concentrations of PC (5、10、20, and 30 mg/L) for 1 h increased the survival rate of PC12 cell in a dose-dependent manner. PC prevented the PC12 cells nuclei from shrinkage, condensation, and cleavage induced by A?_ 25-35 . PC decreased the expression of par-4 mRNA and protein, and increased the expression of bcl-2 mRNA and protein. Conclusion PC can protect PC12 cells from apoptosis induced by A?_ 25-35 in a dose dependent manner. The mechanism of protection is likely related to decreasing the par-4 gene expression and increasing the bcl-2 gene expression.
ABSTRACT
Object To investigate the effects of Heart-benefitting Recipe (HBR) on the histopathological changes of Alzheimer's disease (AD) rats after unilateral amyloid-?_ 25-35 protein (A?_ 25-35 ) injection into the amygdala. Methods The experimental rat models with dementia,spatial learning,and memory impairment were induced by unilateral amyloid ?_ 25-35 protein (A?_ 25-35 ) injection into the amygdala of rats. The spatial learning and memory ability and the function of cholinergic system were observed by the means of Morris water maze and radioligand binding assay. The protein expression of A?_ 1-40 ,AT_8 was estimated with immunohistochemistry. The expression of mRNA related to APP was observed by RT-PCR. Results HBR significantly alleviated the spatial learning and memory impairment induced by A?_ 25-35 and remarkably increased the activity of ChAT and Rt of M-receptor binding sites of these models. The expressions of A?_ 1-40 protein and APP mRNA in cortex and hippocampus of AD rats were decreased remarkably by HBR. The results of immunohistochemistry showed that the expression of hyperphosphorilated tau AT_ 8 in cortex and hippocampus of AD rats was inhibited significantly by HBR. Conclusion HBR can effectively improve the spatial learning and memory impairment,decrease the deposition of A?_ 1-40 ,and alleviate the hyperphos-phorylation of tau of AD rats induced by A?_ 25-35 .
ABSTRACT
Aim To study the effective mechanism of curcumin on abnormal cell cycle and apoptosis of serum-deprived PC12 cells induced by ?-amyloid peptide 25-35(A?25-35).Methods Synchronized PC12 cells were pretreated with 5 ?mol?L-1 Cur for 1 h,and then 25 ?mol?L-1 A?25-35 for 24 h.Protein and mRNA expression of p21,CDK4,E2F1 and bax were detected by RT-PCR and Western blot respectively.Results After synchronized PC12 cells being pretreated with 5 ?mol?L-1 Cur for 1 h,the mRNA and protein expression of p21 gene were increased gradually,while CDK4,E2F1 and bax gene were decreased.Conclusion Cur maybe effects the redistribution of cell cycle and apoptosis of serum-deprived PC12 cells induced by A?25-35,through increasing the mRNA and protein expression of p21,and decreasing the mRNA and protein expression of CDK4,E2F1 and bax gene.
ABSTRACT
AIM To examine the effects of the APP17-mer peptide against A? 25-35 -induced apoptosis and gain some insight into the neuroprotective mechanism of the APP17-mer peptide. METHODS Protective effects of APP17-mer peptide against A? 25-35 -induced apoptosis in SH-SY5Y cell was proved by cell morphology, LM-PCR DNA ladder assay and FCM assay. The antiapoptotic mechanism of APP17-mer peptide was investigated using the MTT assay to measure mitochondrial energy redox state, using the fluorescent probe DCF-DA?Rhodamine 123 to measure relative levels of cellular peroxides and mitochondrial membrane potential and using Western blot for AIF and NF-?B to detect the expression of AIF and NF-?B. RESULTS Damage of cell morphology was ameliorated by pretreating with APP17-mer peptide. The apoptotic rate of the SH-SY5Y cells exposed to A? 25-35 in the presence of APP17-mer peptide decreased from 63.75% to 28.25%. Exposure of SH-SY5Y to A? 25-35 for 48 h resulted in an increase in DCF-DA fluorescence,a decrease in Rhodamine 123 fluorescence and MTT reduction, the results were weakened by pre-incubating with APP17-mer peptide for 30 minutes. Treatment of cells with APP17-mer peptide resulted in a significant attenuation in the expression of AIF and a strong increase in the expression of NF-?B. CONCLUSION APP17-mer is protective against cell apoptosis induced by A? 25-35 by provoking and sustaining upregulation of a key antiapoptotic transcription factor NF-?B, by suppressing oxyradical production and by preserving mitochondrial function and inhibiting the release of apoptotic protein from mitochondria.
ABSTRACT
Aim To study the effect of EA on the injury induced by ?-amyloid protein(A?) in primary cultures of rat hippocampal neurons. Methods The protective effect of EA on A?_25-35 induced neurons injury was observed by LDH release rate, MTT, LSCM and TUNEL. Results A?_25-35 could induced cell death in rat primary hippocampal neurons. Four hours pretreatment with 20 mg?L-1, 40 mg?L-1 EA exerted the protective effect on rat primary hippocampal neurons from A?_25-35 induced injury. Conclusion EA had protective effects against injury induced by A?_25-35 in rat primary hippocampal neurons to some certain,which probably related with decreasing the level of calcium.
ABSTRACT
Aim To determine TAT-tCNTF penetration ability and to investigate the effects of the fusion protein on SH-SY5Y cells against toxicity induced by ?-amyloid peptide 25-35(A?25-35 ).Methods The conjugate(TAT-tCNTF)of TAT(47-57)of HIV-1 and the truncated human CNTF active fragment was genetic engineered and expressed in E.Coli.Immunofluorescence was used to identify cell permeation ability across membrane.MTT assay was used to measure the survival of SH-SY5Y cells injured by A?25-35.And Hoechst 33342/PI double staining was used to observe the morphology of cell apoptosis and necrosis.LDH was measured by spectrophotometric method.Results The expression vector of pBV220-TAT-tCNTF was constructed successfully.Western blot showed the recombinant fusion protein could bind specifically with CNTF antibody.The immunofluorescence assay clearly demonstrated that TAT-tCNTF did penatrate into the cells while little rhCNTF pass across the cells.Double staining and LDH release assay demonstrated that TAT-tCNTF could promote significantly the survival of the cells.Conclusions TAT-tCNTF with high activities and effective transmembrane ability is obtained for the first time.The fusion protein protects SH-SY5Y cells from death after A?25-35 exposure.